The detection of specified antigens (defined as a substance whose introduction into an animal stimulates the production of antibodies capable of reacting specifically therewith), haptens (a substance requiring additional accessory materials before its introduction into an animal stimulates the production of antibodies specific therefor), and the like substances (hereinafter collectively referred to as ligands) in body fluids such as blood, sputum, urine and the like has in recent years become of utmost importance in both the research and clinical environments. The detection of ligands, particularly antigens or antibodies capable of specifically combining therewith (hereinafter collectively termed anti-ligands, which term is also meant to include antibody fragments such as F(ab), F(ab)'.sub.2 etc.) can often be related to various disease states and consequently is extremely useful in diagnosis, in gaining basic understandings concerning the genesis of disease, and in monitoring the effectiveness of therapies therefor. The high level of specificity between ligands and antiligands has permitted the development of an entire class of assays typically referred to as immunoassays which are based on this immunological reaction or coupling therebetween. As will be readily appreciated by those skilled in the art, there are a great many ways in which immunoassays may be formatted depending upon whether the assays are to be performed simultaneously, in a forward or reverse direction, competitively, non-competitively, or in a homogeneous or heterogeneous manner as those terms and methods are readily understood and commonly practiced in the art.
One characteristic common to many of the foregoing types of assays, particularly those of the heterogeneous character, is the attachment of one of the components of a ligand-anti-ligand pair to a solid phase such as a plastic or glass surface, the surface of a microtiter tray, microparticle, paddle, etc. This solid phase or surface is typically inserted into the aqueous phase so as to allow detection of the aqueous sample complementary component of the ligand-anti-ligand pair by virtue of an immunological reaction occurring therebetween. Thus, following such a reaction, the aqueous component also becomes immobilized thereby allowing the substantial removal of both unreacted aqueous components and the concommitant associated background "noise" levels whose presence is generally deleterious to sensitivity.
Although a variety of schemes have been developed for the immobilization of ligand or anti-ligands onto a variety of solid phase surfaces, none have proved entirely satisfactory, often for reasons not altogether clear. Generally, attachment of the ligand or anti-ligand is preceded by some form of chemical manipulation such as the attachment of suitable groups for subsequent linkage onto the surface whereby washing steps or other mechanical manipulations do not result in their displacement. These chemical manipulations are, however, often deleterious to the immunological reactivity of the component and in certain circumstances, can actually render the component inactive thereby destroying its usefulness. Further, actual attachment of these ligand or anti-ligand components to the surface, whether by chemical or physical means, takes place in an unknown manner. Contemporary beliefs hold the components may actually attach in a spectrum of positions ranging from complete steric hindrance of the binding site, thereby preventing or significantly reducing reactivity, to unhindered access of the binding site. As may be expected, sensitivity of the assay declines with increasing levels of steric hindrance and loss of reactivity.
It is an object of the present invention to provide new methods for attaching a component of a ligand-anti-ligand pair which are less sensitive to steric orientation effects associated with prior methods and are accordingly more conveniently manufactured.
In one class of instruments useful with immunoassays such as that of Louis A. Kamentsky described in U.S. Ser. No. 455,765, now U.S. Pat. No. 4,487,839, entitled "Immunoassay Methods Employing Patterns For The Detection Of Soluble And Cell Surface Antigens", soluble antigens are reacted with antibodies which have been attached to a surface in a particular pattern. The pattern can be generally characterized as a repeating and alternating presence and absence of immunological reactivity. The repeated examination of areas having an immunological reaction and areas having an absence of such immunological reaction allows the subtraction of the latter area or background level from the test area and results in an instrument of great sensitivity. Of great importance to such an instrument is the accuracy and repeatability to which the anti-ligands are spatially resolved or patterned on the solid phase surface. Methods presently available to date are not sufficiently accurate when practiced on a commercial manufacturing scale to permit the production of such patterns in an acceptable and repeatable manner. Further, conventional methods for attaching anti-ligands or ligands, particularly immunoglobulins, to solid phase surfaces often disadvantageously results in short shelflifes thereby exacerbating production, shipment and storage difficulties.
It is another object of the present invention to provide methods which increase the shelf-life of the resultant solid phase component.
It is yet a further object of the present invention to provide methods of attachment whereby patterns may be produced having great repeatable accuracy and sharp lines of demarcation.
It is yet another object of the present invention to provide apparatus and methods useful in a variety of immunoassays, particularly with those based on pattern detections, which also permits commercial manufacture of such solid phase components in a repeatable and economical manner.